Antiviral methods using fragments of human rhinovirus receptor (ICAM-1)

ABSTRACT

Antiviral methods comprising contacting human rhinovirus with tICAM(453).

This application is a division of copending application U.S. Ser. No.08/316,885, filed Sep. 30, 1994, which is a continuation of U.S. Ser.No. 08/103,303 filed Aug. 6, 1993, now abandoned, which is acontinuation of U.S. Ser. No. 07/631,313 filed Dec. 20, 1990, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 07/556,238filed Jul. 20, 1990, now abandoned, and a continuation-in-part of U.S.Ser. No. 07/390,662 filed Aug. 10, 1989, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/239,571 filed Sep. 1, 1988, nowabandoned, and a continuation-in-part of U.S. Ser. No. 07/262,428 filedOct. 25, 1988, now abandoned, which is also a continuation-in-part ofU.S. Ser. No. 07/239,571.

BACKGROUND OF THE INVENTION

The present invention relates to novel truncated forms of intercellularadhesion molecule (ICAM), designated “tICAMs”, which effectively bind tohuman rhinovirus (HRV) and to lymphocyte-function associated antigen-1(LFA-1). The present invention also pertains to DNA sequences coding forvarious tICAMs and to methods for preventing or amelio-rating infectionand inflammation using said tICAMs.

Human rhinoviruses, the major causative agent of the common cold, belongto the picornavirus family. The three-dimensional structure of severalrhinovirus serotypes have now been determined to atomic resolution byRossmann, M. G., E. Arnold, J. W. Erickson, E. W. Frankenberger, P. J.Griffith, H. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R.Rueckert, B. Sherry, and G. Vriend, Nature (1985) 317:145-153; Kim, S.,T. J. Smith, M. M. Chapman, M. G. Rossmann, D. C. Pevear, F. J. Dutko,P. J. Felock, G. D. Diana, and M. A. McKinlay, J. Mol. Biol. (1990)210:91-111. The virion is composed of a protein capsid of 60 protomericunits, consisting of the four protein subunits VP1-4, surrounding an RNAgenome. Each of the 60 protomeric units possesses a recessed “canyon”that is believed to contain the site that binds to the receptor on thetarget cell surface [reviewed in Rossmann, M. G., J. Biol. Chem. (1989)264:14587-14590]. The dimensions of the canyon are such that it is toosmall to admit the combining site of an antibody but is apparently largeenough to admit the virus-binding site of the receptor.

In order to infect host cells, viruses must bind to and then enter cellsto initiate an infection. Since 1959, evidence has accumulated in theliterature indicating that the presence of specific binding sites(receptors) on host cells could be a major determinant of tissue tropismof certain viruses. [Holland, J. J., and L. C. McLaren, “The mammaliancell-virus relationship. II. Absorption, reception, and eclipse ofpoliovirus by HeLa cells,” J. Exp. Med. 109:487-504 (1959); Holland, J.J., “Receptor affinities as major determinants of enterovirus tissuetropisms in humans,” Virology 15:312-326 (1961)]. Among picornavirusessuch as poliovirus, Coxsackie virus, and rhinoviruses, specific bindingto host cells has been demonstrated. By competition experiments, it hasbeen demonstrated that some of these receptors are distinct from oneanother in that the saturation of the receptor of one virus had noeffect on the binding of a second virus. [Lonberg-Holm, K., R. L.Crowell, and L. Philipson. “Unrelated animal viruses share receptors,”Nature 259:679-681 (1976)].

Rhinoviruses can be classified according to the host cell receptor towhich they bind. Tomassini, J. E. and R. J. Colonno, “Isolation of areceptor protein involved in attachment of human rhinoviruses,” J.Virol. 58:290 (1986) reported the isolation of a receptor proteininvolved in the cell attachment of HRV. Approximately 90% of the morethan 115 serotypes of rhinoviruses, as well as several types ofCoxsackie A virus, bind to a single common receptor termed the “major”human rhinovirus receptor (HRR) [Abraham, G., and R. J. Colonno, “Manyrhinovirus serotypes share the same cellular receptor,” J. Virol.51:340-345 (1984)]; the remaining 10% bind to one or more other cellreceptors.

The major human rhinovirus receptor has been transfected, identified,purified, and reconstituted as described in co-pending U.S. patentapplications Ser. Nos. 07/262,428 and 07/262,570, both filed Oct. 25,1988. Greve, J. M., G. Davis, A. M. Meyer, C. P. Forte, S. C. Yost, C.W. Marlor, M. E. Kamarck, and A. McClelland, “The major human rhinovirusreceptor is ICAM-1,” Cell 56:839 (1989), identified the major HRR as aglycoprotein with an apparent molecular mass of 95 kD and having anamino acid sequence essentially identical to that deduced from thenucleotide sequence of a previously described cell surface protein namedintercellular adhesion molecule (ICAM-1). ICAM-1 had first beenidentified based on its role in adhesion of leukocytes to endothelialcells [Rothlein, R., et al., J. Immunol. 137:1270-1274 (1986); see alsoSimmons, D., M. W. Makgoba, and B. Seed, Nature (1988) 331:624;Staunton, D. E., S. D. Marlin, C. Stratowa, M. L. Dustin, and T. A.Springer, “Primary structure of ICAM-1 demonstrates interaction betweenmembers of the immunoglobulin and integrin supergene families,” Cell(1988) 52:925-933]. Induction of ICAM-1 expression by cytokines duringthe inflammatory response may regulate leukocyte localization toinflammatory sites. Subsequently, Staunton, D. E., et al., Cell 56:849(1989) confirmed that ICAM-1 is the major cell surface receptor for HRV.See also Staunton, D. E., M. L. Dustin, H. P. Erickson, and T. A.Springer, “The arrangement of the immunoglobulin-like domains of ICAM-1and the binding sites for LFA-1 and rhinovirus,” Cell (1990) 61:243-254.The precise extent of the virus-binding site on ICAM-1 remains to bedetermined, although results from mouse-human chimeras and site-directedmutagenesis indicate that the two N-terminal domains play a major rolein virus binding [Staunton, et al., Cell (1990) 61:243-254], and a modelhas been developed for the interaction of the N-terminal domain ofICAM-1with HRV14 [Giranda, V. L., M. S. Chapman, and M. G. Rossmann,Proteins (1990) 7:227-233].

European Patent Application 0 289 949 describes membrane-associatedICAM-1, which mediates attachment of many cell types, includingendothelial cells, to leukocytes expressing lymphocyte functionassociated molecule-1 (LFA-1; CD18/CD11a, a member of the beta-2integrin family). This patent application provides a discussion of theprior research in the field of intercellular adhesion molecules.

Heterotypic binding of LFA-1 to ICAM-1 mediates cellular adhesion ofdiverse cell types and is important in a broad range of immuneinteractions [Marlin, et al., Cell (1987) 51:813-819]. ICAM-1 also bindsto MAC-1 (CD18/CD11b), another beta-2 integrin, but not to p150/95(CD18/CD11c) [Staunton, D. E., S. D. Marlin, C. Stratowa, M. L. Dustin,and T. A. Springer, Cell (1988) 52:925-933]. MAC-1 and p150/95 differfrom LFA-1 by their alpha subunit. Although minimal peptide recognitionsites have been identified for many other integrins, the recognitionsite for LFA-1 on ICAM-1 remains obscure. Staunton, et al., Cell (1990)61:243-254 have reported that a transmembrane form of the first twodomains of ICAM-1 retains some LFA-1-binding activity and that a numberof mutations in the first two domains of the full-length molecule causereductions in LFA-1-binding activity.

The primary structure of ICAM-1 is homologous to two other cellularadhesion molecules: neural cell adhesion molecule (NCAM) andmyelin-associated glycoprotein (MAG). This suggests that ICAM-1 is amember of the immunoglobulin supergene family [Simmons, et al., Nature(1988) 331:624-627; Staunton et al., Cell (1988) 52:925-933]. The cDNAsequences are described in the above-referenced papers by Simmons et al.and Staunton et al., from which the amino acid sequence of ICAM-1 hasbeen deduced.

ICAM-1 is an integral membrane protein 505 amino acids long and has: i)five immunoglobulin-like extra-cellular domains at the amino-terminal(extracellular) end (designated domain 1 [amino acid residues 1-88],domain 2 [89-185], domain 3 [186-284], domain 4 [285-385], and domain 5[386-453] [Staunton, et al., Cell (1988) 52:925-933]); ii) a hydrophobictransmembrane domain (454-477); and iii) a short cytoplasmic domain atthe carboxy-terminal end (478-505). Electron microscopy has indicatedthat ICAM-1 is a highly elongated molecule [Staunton, et al., Cell(1990) 61:243-254].

Several approaches to decreasing infectivity of viruses in general, andof HRV in particular, have been pursued including: i) developingantibody to the cell surface receptor for use in blocking viral bindingto the cell; ii) using interferon to promote an anti-viral state in hostcells; iii) developing various agents to inhibit viral replication; iv)developing antibodies to viral capsid proteins/peptides; and v) blockingviral infection with isolated cell surface receptor protein thatspecifically blocks the viral binding domain of the cell surfacereceptor.

In 1985, the isolation of a monoclonal antibody that appeared to bedirected against the major rhinovirus receptor was described. [Colonno,R. J., P. L. Callahan, and W. J. Long, “Isolation of a monoclonalantibody that blocks attachment of the major group of humanrhinoviruses,” J. Virol. 57:7-12 (1986)]. This monoclonal inhibitedinfection of cells with the appropriate serotypes of rhinovirus and itinhibited binding of radiolabeled rhinovirus to cells. Colonno et al.subsequently reported that the monoclonal antibody bound to a proteinwith an apparent molecular weight of 90 kD [Tomassini, et al., J. Virol.(1986) 58:290-295]. This monoclonal antibody has been utilized inclinical trials with primates and humans and is understood to providesome protection against rhinovirus infection.

There are several other reports of attempts at therapeutic interventionin rhinovirus infections. Intranasal application of interferon in humanshas been attempted. [Douglas, R. M. et al., “Prophylactic efficacy ofintranasal alpha2-interferon against rhinovirus infections in the familysetting,” N. Eng. J. Med. 314:65-75 (1986)]. In this case, significantreduction in the severity of the infection was found, althoughnosebleeds were observed as a side-effect. Also, several analogs ofdisoxaril (“WIN” compounds) that reduce the infectivity of a number ofpicornaviruses (with widely varying effectiveness, depending on theserotype) have been tested in tissue culture and in some animal models[Fox, M. P., M. J. Otto, and M. A. McKinlay, Antimicrob. Ag. andChemotherapy (1986) 30:110-116]. These compounds appear to inhibitreplication at a step subsequent to receptor binding, probably at somestep of virus uncoating. The atomic coordinates of the binding sites ofthese compounds within the viral capsid of the serotype HRV14 have beendetermined by x-ray crystallography, and are located in a hydrophobicpocket present in each protomeric unit of the capsid [Smith, T. J., etal., “The site of attachment in human rhinovirus 14 for antiviral agentsthat inhibit uncoating,” Science (1986) 233:1286-1293]. The specificfunction of the binding pocket, if any, is unknown, but drug-resistantmutants with a single amino acid interchange in this region arise athigh frequency and are viable [Badger, J., et al., “Structural analysisof a series of antiviral agents complexed with human rhinovirus 14,”PNAS 85:3304-3308 (1988); see also Dearden et al., Arch. Virol. (1989)109:71]. This result calls into question the efficacy of such compoundsas drugs. The production of anti-peptide antibodies in rabbits has beenreported using peptides derived from amino acid sequences of the viralcapsid proteins that line the “receptor canyon” of HRV14 [McCray, J.,and G. Werner, “Different rhinovirus serotypes neutralized byantipeptide antibodies,” Nature (1987) 329:736-738]. While the titers ofthese sera are quite low, cross-serotype protection of cells in tissueculture from rhinovirus infection was demonstrated, raising thepossibility of a vaccine.

It is an object of the present invention to provide an HRV receptorprotein having the property of blocking HRV infection. Given the highaffinity the virus has for its receptor, a therapeutic agent effectiveagainst HRV infection is the receptor itself, or more specifically, thevirus-binding domain of the receptor. A protein, protein fragment, orpeptide that comprises the virus-binding domain can block the ability ofvirus to bind to host cells by occupying (blocking) the receptor bindingcleft on the virus. Furthermore, since such a molecule makes some or allof the molecular contacts with the virus capsid that the receptor does,virus mutations that adversely affect binding of the molecule alsoadversely affect binding of the receptor, and thus are deleterious orlethal for the virus; therefore, the likelihood of drug-resistantmutants is very low.

Using this approach, Greve, et al., Cell (1989) 56:879, reported thatpurified transmembrane ICAM-1 (tmICAM-1) could bind to rhinovirus HRV3in vitro. Other results with HRV2, HRV3, and HRV14 demonstrate apositive correlation between the ability to bind to rhinovirus and theability to neutralize rhinovirus. Results using HRV14 and HRV2demonstrate a positive correlation between the receptor class of thevirus and the ability to bind to tmICAM-1 in vitro. That is, ICAM-1,being the major receptor, can bind to HRV3, HRV14, and other majorreceptor serotypes and neutralize them, while it does not bind orneutralize HRV2, a minor receptor serotype. Further studies, usingpurified tmICAM-1, demonstrate that it effectively inhibits rhinovirusinfectivity in a plaque-reduction assay when the rhinovirus ispretreated with tmICAM-1 (50% reduction of titer at 10 nM receptor andone log reduction of titer at 100 nM receptor protein). These data wereconsistent with the affinity of rhinovirus for ICAM-1 of HeLa cells,which has an apparent dissociation constant of 10 nM, and indicates adirect relationship between the ability of the receptor to bind to thevirus and to neutralize the virus.

The ICAM of the prior art is an insoluble molecule which is solubilizedfrom cell membranes by lysing the cells in a non-ionic detergent.Because large-scale production of tmICAM-1 is not presently economicallyfeasible, and because maintenance of tmICAM-1 in an active form requiresthe use of detergents, alternate soluble forms of receptor proteinsuitable for use as a rhinovirus inhibitor are desirable. See generallycopending applications U.S. Ser. Nos. 07/130,378; 07/239,570;07/239,571; 07/262,428; 262,570; 07/390,662; and 07/556,238, allincorporated herein by reference.

U.S. Ser. No. 07/130,378 (filed Dec. 8, 1987) and its CIP applicationU.S. Ser. No. 07/262,570 are directed to transfected non-human celllines which express the major human rhino-virus receptor (HRR), and tothe identification of HRR as intercellular adhesion molecule (ICAM).

U.S. Ser. No. 07/301,192 (filed Jan. 24, 1989) and its CIP applicationsU.S. Ser. No. 07/445,951 (abandoned) and U.S. Ser. No. 07/449,356 aredirected to a naturally-occurring soluble ICAM (sICAM) related to butdistinct from tmICAM in that said sICAM lacks the amino acids spanningthe hydrophobic transmembrane region and the carboxy-terminalcytoplasmic region; in addition this sICAM has a novel sequence of 11amino acids at its C-terminus.

Parent application U.S. Ser. No. 07/239,571 (filed Sep. 1, 1988) and itsCIP applications U.S. Ser. Nos. 07/262,428 and 07/390,662 are directedto the use of detergent-complexed tmICAM as an inhibitor of HRVinfectivity, using the non-ionic detergent complex to maintain thetransmembrane protein in solution. These cases are also directed totruncated forms of ICAM comprising one or more of the extracellulardomains I, II, and III, IV and V of tmICAM, which truncated forms do notrequire the presence of non-ionic detergent for solubilization.

Parent application U.S. Ser. No. 07/556,238 (filed Jul. 20, 1990) isdirected to multimeric configurations and forms of tmICAM-1 and tICAM-1with improved ability to prevent HRV infection. When tmICAM-1,tICAM(453), or tICAM(185) is first adsorbed on any of a number ofinsoluble supports, such as nitrocellulose, PVDF (polyvinylidenedifluoride), or DEAE (diethylaminoethyl) membranes, and then incubatedwith radioactive HRV, the virus-binding activity of tICAM(453) becomescomparable to that of tmICAM-1. This binding of multimeric tICAM(453) orof multimeric tICAM(185) to HRV has the same properties as the bindingof HRV to ICAM-1 on HeLa cells: it is inhibited by anti-ICAM-1monoclonal antibodies, it is specific for HRV of the major receptorgroup, and it has the same temperature-dependence pattern as the bindingof HRV to cells (i.e., binds well at 37° C. and undetectably at 4° C.).

The present application contains further data demonstrating theproperties and efficacy of various forms of tICAM.

SUMMARY OF THE INVENTION

The present invention provides truncated forms of ICAM-1 designatedtICAM-1 which are soluble without the addition of a detergent.

The present invention also provides purified and isolated tICAM-1, orfunctional derivatives thereof, substantially free of naturalcontaminants.

The present invention further provides a purified and isolated DNAsequence encoding tICAMs as well as host cells encoding said sequences.

The present invention provides a method of recovering tICAMs insubstantially pure form comprising the steps of cloning a genome codingfor the desired tICAM form in a suitable expression vector in a suitablehost.

Also provided by the invention are novel pharmaceutical compositionscomprising a pharmaceutically acceptable solvent, diluent, adjuvant orcarrier, and as the active ingredient, an effective amount of apolypeptide characterized by having HRV binding activity and reductionof virus infectivity.

The present invention includes monoclonal antibodies against ICAM-1 andtICAMs, and hybridoma cell lines capable of producing such monoclonalantibodies.

This invention further includes the therapeutic use of antibodiesspecifically directed to tICAMs to decrease cell adhesion mediated byICAM-1 and LFA-1.

The invention further includes a method of inhibiting LFA-1 and ICAM-1interaction comprising the step of contacting LFA-1-containing cellswith a tICAM or a functional derivative thereof.

A further aspect of the invention is use of fragments, functionaldomains or analogs of LFA-1 to disrupt interactions between HRR and HRVand thereby treat HRV infections.

This invention further includes a method for substantially reducinginfection by HRV of the major receptor group, comprising the step ofcontacting the virus with tICAM-1 or a functional derivative thereof.

This invention further includes a method of diagnosis of the presenceand location of an LFA-1-expressing tumor cell.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description thereof whichincludes numerous illustrative examples of the practice of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the domains included inseveral representative tICAM constructs of the present invention.

FIG. 2 shows secretion of soluble ICAM-1 proteins. A. Diagram ofprogressively truncated forms of ICAM-1 used in transfectionexperiments. Crosshatched region indicates signal sequence and filledregion indicates the transmembrane region. B. Fluorograph of³⁵S-cysteine-labelled products secreted by COS cells analyzed bySDS-PAGE; loading of lanes containing tICAM(283) and tICAM(88) is10-fold higher than other lanes. C. Silver-stained gel of purifiedtICAM(453) and tICAM(185) produced by CHO cell transfectants.

FIG. 3 shows circular dichroism spectra of IgG and ICAM-1 proteins. A:c92.5 IgG; B: ICAM-1; C: tICAM(453); D: tICAM(185). The spectra forc92.5 IgG, tICAM(453), and tICAM(185) were collected in 20 mM sodiumphosphate buffer (pH 7.5) and the spectrum for ICAM-1 was collected in0.1% octylglucoside/150 mM NaCl/10 mM sodium phosphate (pH 7.5). Thedata were averaged from 5 repetitive scans, buffer-subtracted andsmoothed.

FIG. 4 shows inhibition of ICAM-1/LFA-1-mediated cell adhesion bysoluble ICAM-1. JY cells were preincubated with various concentrationsof tICAM(453) (filled circles) or tICAM(185) (open squares) for 30 minand then allowed to bind to ICAM-1-coated plates for 60 min at 37 C. Thebinding of JY cells is >90% inhibited by monoclonal antibody c78.4 toICAM-1 and IOT 16 (AMAC Inc.) to LFA-1.

DETAILED DESCRIPTION

As used herein, the following abbreviations and terms include, but arenot necessarily limited to, the following definitions:

Abbreviation Definition ICAM Intercellular adhesion molecule - may beused to denote both full length (transmembrane) and truncated forms ofthe protein. ICAM-1 Intercellular adhesion molecule-1, also known astmICAM-1 and HRR; denoting the full- length transmembrane protein.tmICAM-1 Transmembrane intercellular adhesion molecule-1, also known asICAM-1 and HRR; requires., e.g., detergent conditions to be solubilized.HRR Human rhinovirus receptor, also known as ICAM-1 and tmICAM-1.sICAM-1 A naturally-occurring soluble truncated form of ICAM-1 havingboth the hydrophobic transmembrane domain and the carboxyl-terminalcytoplasmic domain of ICAM-1 deleted; consists of amino acids 1-442 ofICAM-1 plus 11 novel amino acids. tICAMs Truncated intracellularadhesion molecules; soluble non-transmembrane ICAMs lacking thehydrophobic transmembrane domain and the carboxyl-terminal cytoplasmicdomain of ICAM-1. tICAM(453) Truncated form of ICAM comprising theentire extracellular amino-terminal domain of ICAM (domains I-V, aminoacid residues 1-453). tICAM(283) Truncated form of ICAM comprisingdomains I, II, and III (amino acid residues 1-283). tICAM(185) Truncatedform of ICAM comprising domains I and II (amino acid residues 1-185).tICAM(88) Truncated form of ICAM comprising domain I (amino acidresidues 1-88).

The ICAM-1 terminology has been used although it is now recognized thatthe terms HRR and ICAM-1 are interchangeable.

“Multimerization” and “multimeric” include, but are not limited todimerization and dimeric, and include any multimeric configuration ofthe ICAM-1 molecule, or fragment thereof, that is effective in reducingviral binding and infectivity.

“Transmembrane” generally means forms of the ICAM-1 protein moleculewhich possess a hydrophobic membrane-spanning sequence and which aremembrane-bound.

“Non-transmembrane” generally means soluble forms of the ICAM-1 proteinincluding truncated forms of the protein that, rather than beingmembrane bound, are secreted into the cell culture medium as solubleproteins, as well as transmembrane forms that have been solubilized fromcell membranes by lysing cells in non-ionic detergent.

“Truncated” generally includes any protein form that is less than thefull-length transmembrane form of ICAM.

“Form” is generally used herein to distinguish among full-length andpartial-length ICAM forms; whereas “configuration” is generally used todistinguish among monomeric, dimeric, and multimeric configurations ofpossible ICAM forms.

All forms and configurations of the ICAM-1 molecule, whether full lengthor a fragment thereof, including muteins, whether monomeric ormultimeric, may be fully or partially glycosylated, or completelyunglycosylated, as long as the molecule remains effective in reducingviral binding and infectivity.

“Ligand” is generally used herein to include anything capable of bindingto at least one of any of the forms and configurations of ICAM andincludes, but is not limited to, HRV, other viruses that bind to the“major” group human rhinovirus receptor, LFA-1, and Plasmodiumfalciparum (the causative agent of malaria).

“Human rhinovirus” or “HRV” generally includes all human serotypes ofhuman rhinovirus as catalogued in Hamparian, V., et al., Virol. (1987)159:191-192.

The sequence of amino acid residues in a peptide is designated inaccordance with standard nomenclature such as that given in Lehninger'sBiochemistry (Worth Publishers, New York, 1970).

Full-length ICAM-1, also known as human rhinovirus receptor (HRR), istermed transmembrane ICAM (tmICAM-1). The present invention providesnon-transmembrane truncated forms of ICAM-1 designated tICAM-1, i.e.,ICAM substantially without the carboxyl-terminal cytoplasmic domain andwithout the hydrophobic transmembrane domain, which are soluble withoutthe addition of a detergent. Non-transmembrane forms of ICAM can includefunctional derivatives of ICAM and mutein forms of tICAM. Preferredembodiments are tICAM(453), tICAM(283), tICAM(185), and tICAM(88).

The present invention further provides purified and isolated tICAMs, orfunctional derivatives thereof, substantially free of naturalcontaminants. tICAMs can be obtained from transfected cells, e.g., CHOcells, and are characterized by being soluble in the absence of nonionicdetergents and being the translation product defined by novel DNAoligomers. These products have the advantage of being secreted fromcells in a soluble form and of not being immunogenic. These truncatedproducts differ from the natural insoluble product in that the truncatedsoluble products do not contain the membrane-spanning and cytoplasmicdomains present in the insoluble form.

The present invention provides purified and isolated DNA sequencesencoding tICAMs as well as host cells encoding said sequences. Thenucleotide sequences coding for tICAMs can be contained in vectors, suchas plasmids, and the vectors can be introduced into host cells, forexample eukaryotic or prokaryotic cells. Examples of suitable eukaryoticcells are mammalian cells, e.g. Chinese hamster ovary cells, and yeastcells. Examples of suitable prokaryotic cells are, e.g, E. coli.Eukaryotic cells are preferred; mammalian cells are particularlypreferred.

Also provided by the invention are novel pharmaceutical compositionscomprising a pharmaceutically acceptable solvent, diluent, adjuvant orcarrier, and as the active ingredient, an effective amount of apolypeptide characterized by having HRV-binding activity and reductionof virus infectivity.

The invention further provides novel pharmaceutical compositionscomprising a pharmaceutically acceptable solvent, diluent, adjuvant orcarrier, and as the active ingredient, an effective amount of apolypeptide characterized by having LFA-1-binding activity and reductionof LFA-1 activity.

The present invention further provides methods of recovering a tICAM insubstantially pure form comprising the steps of cloning a gene codingfor the desired tICAM form in a suitable expression vector in a suitablehost. The secretion of a soluble protein eliminates the problemsassociated with production and purification of an insoluble,cell-membrane-bound protein, since cell lysis is not required and thuscontinuous culture can be employed along with simplified procedures forpurification and isolation of the tICAMs. The protein may be purifiedusing an immunoaffinity column, lectin or wheat germ agglutinin column.Other purification steps may include sizing chromatography, ionchromatography, and gel electrophoresis. The antibody capable of bindingto tICAM is selected from antibodies against ICAM-1 (tmICAM or HRR) orfragments thereof.

The present invention includes polyclonal antibodies against tICAM. Fora method for producing peptide antisera see Green et al., Cell28:477-487 (1982).

The invention also includes hybridoma cell lines capable of producingmonoclonal antibodies to tICAMs, and the monoclonal antibodies producedby said cell lines.

This invention further includes the therapeutic use of antibodiesspecifically directed to tICAMs to decrease cell adhesion mediated byICAM and LFA-1.

Pharmaceutical preparations of proteins, protein fragments, functionaldomains, and analogs have an application in a plurality of diseases.Since the various forms of ICAM are ligands for both LFA-1 and HRV, theymay also be used to block tmICAM interaction with LFA-1, which iscritical to many cell adhesion processes involved in the immunologicalresponse. This method of inhibition of ICAM-1-mediated adhesion hasapplication in such disease states as inflammation and graft rejection,and for suppression of LFA-1-expressing tumor cells and other processesinvolving cell adhesion.

A further aspect of the invention is use of fragments, functionaldomains or analogs of LFA-1 to disrupt interactions between HRR andrhinovirus and thereby treat rhinovirus infections.

This invention further includes a method of diagnosis of the presenceand location of an LFA-1-expressing tumor cell.

This invention further includes a method for substantially reducinginfection by other pathogens of the major HRR group, e.g., Coxsackievirus and Plasmodium falciparum, comprising the step of contacting thepathogen with one or more tICAMs or functional derivatives thereof.

The following examples describe preparation and characterization ofrepresentative compounds of the present invention and representativecompositions containing said compounds. As used hereinabove and belowunless expressly stated to the contrary, all temperatures andtemperature ranges refer to the centrigrade system; the term percent or(%) refers to weight percent; and the term mole and moles refer to grammoles.

EXAMPLE 1 Growth, Purification and Assay of Rhinoviruses

Rhinoviruses were grown, purified, and assayed essentially as describedin Abraham, G., et al., J. Virol. (1984) 51:340 and Greve, et al., Cell,(1989) 56:839. The serotypes chosen for these studies include HRV14, thestandard in the field, and HRV3, which has an approximately 10-foldhigher affinity for ICAM than does HRV14. HRV2, which binds to the“minor” receptor rather than the “major” receptor, was used as anegative control.

Rhinoviruses HRV2, HRV3, and HRV14 were obtained from the American TypeCulture Collection, plaque purified, and isolated from lysates ofinfected HeLa-S3 cells. Purified rhinovirus was prepared by polyethyleneglycol precipitation and sucrose density gradient sedimentation. Viralpurity was assessed by SDS-PAGE analysis of capsid proteins and byelectron microscopy. Infectivity was quantified by a limiting-dilutioninfectivity assay scoring for cytopathic effect, essentially asdescribed by Minor, P. D., “Growth, assay and purification ofpicornaviruses,” in Virology: A Practical Approach, B. W. J. May, ed.(Oxford, IRL Press, 1985), pp. 25-41.

EXAMPLE 2 Production and Isolation of Monoclonal Antibodies to ICAM-1

BALB/cByJ female mice were immunized by intraperitoneal injection of 10⁷intact HeLa cells in 0.5 ml of phosphate-buffered saline (PBS) threetimes at 3-week intervals. Two weeks later the mice were bled andaliquots of serum were tested for protective effects against HRV14infection of HeLa cells. Positive mice were boosted by a final injectionof 10⁷ HeLa cells, and 3 days later spleen cells were fused toP3X63-Ag8.653 myeloma cells [Galfre, et al., Nature (1977) 266:550-552]produce a total of approximately 700 hybridoma-containing wells. Eachwell was tested by incubating 3×10⁴ HeLa cells in 96-well plates with100 μl of supernatant for 1 hour at 37° C.; the cells were then washedwith PBS, and a sufficient amount of HRV14 was added to give completecytopathic effect in 24-36 hours. Wells that were positive (protectedfrom infection) were scored at 36 hours.

Cells were removed from wells which scored positive in the first screenand cloned by limiting dilution in 96-well microtiter plates.Supernatants from these wells were tested in the cell protection assayand positive wells were again identified. Further clonings wereperformed until all of the hybridoma-containing wells were positiveindicating a clonal population had been obtained. Four cloned celllines, and their corresponding antibodies, were obtained and weredesignated c78.1A, c78.2A, c78.4A, c78.5A. An additional two cell lineswere obtained by hyperimmunizing mice with L-cells transfected withhuman genomic DNA comprising HRR and were designated c92.1A and c92.5A.

C92.1A was deposited on Nov. 19, 1987 with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 and wasdesignated HB 9594.

EXAMPLE 3 cDNA Constructions

ICAM-1 cDNAs encoding soluble forms were constructed by using polymerasechain reaction (PCR) [Saiki, et al., Science (1985) 230:1350-54] toinsert stop codons within the reading frame of the ICAM-1 cDNA pHRR-2,[Greve, et al., Cell (1989) 56:839-847]. The plasmid DNA was digestedwith EcoR1 to excise the ICAM-1 insert and treated with alkalinephosphatase to prevent recircularization of the vector in subsequentligation steps. 10 ng of template DNA was subjected to 10 cycles of PCRamplification using the 5′ oligonucleotide primerGGAATTCAAGCTTCTCAGCCTCGCTATGGCTCCCAGCAGCCCCCGGCCC [SEQ ID NO:1] and the3′ oligonucleotide primers GGAATTCCTGCAGTCACTCATACCGGGGGGAGAGCACATT [SEQID NO:3] [for tICAM (453)], TTCTAGAGGATCCTCAAAAGCTGTAGATGGTCACTGTCTG[SEQ ID NO:3] [for tICAM (283)], TTCTAGAGGATCCTCAAAAGGTCTGGAGCTGGTAGGGGG[SEQ ID NO:4] [for tICAM (185)], andTTCTAGAGGATCCTCACCGTTCTGGAGTCCAGTACACGG [SEQ ID NO:5] [for tICAM(88)].The PCR reaction products were digested with EcoR1 [tICAM(453)] orEcoR1and BAMH1 [tICAM(283), tICAM(185), and tICAM(88)] and cloned intothe polylinker site of Bluescript SK+ (Stratagene). In this manner, stopcodons were inserted immediately before the first residue of thetransmembrane domain, at the predicted ends of domains I+II+III, domainsI+II, and domain I to produce a series of progressively truncatedproteins (FIG. 2A). Clones containing the desired inserts were verifiedby restriction analysis and DNA sequencing.

The inserts were excised by digestion with HindIII and XbaI, insertedinto the expression vector CDM8 [Seed, B., Nature (1987) 239:840-42],transfected into COS cells for transient expression, and co-transfectedwith pSV2-DHFR into CHO cells for establishment of stable cell lines.For transient expression, COS cells were transfected by the DEAE-dextranmethod [Kingston, R. E., in Current Protocols in Molecular Biology, F.M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.Smith, and K. Struhl, eds., pp. 9.0.1-6] and pulse-labeled 72 hoursafter transfection with [³⁵S]-cysteine for 18 hours; culturesupernatants were then immunoprecipitated with c78.4 IgG-SEPHAROSE®(high molecular weight filtration gel, Pharmacia, Piscataway, N.J.) andanalyzed by SDS-PAGE [Greve, et al., Cell (1989) 56:839-847]. Stable CHOtransfectants were obtained by cotransfection of ICAM-1 cDNAs withpSV2-DHFR into dihydrofolate reductase-deficient CHO cells by thecalcium phosphate method or by electroporation [Bebbington andHentschel, in DNA Cloning—A Practical Approach, Vol. 3, D. M. Glover,ed. (IRL Press, Oxford, 1987), pp. 163-188].

This experiment indicated that the entire extracellular domain,tICAM(453), and the two N-terminal domains, tICAM(185), were efficientlysecreted from transfected COS cells as species of 80 kD and 43 kD,respectively (FIG. 2B). The expression of domains I+II+III [tICAM(283)]was approximately 10-fold lower than the above-mentioned fragments andthe secreted protein was more heterogeneous in mobility on SDS-PAGE.Expression of domain I [tICAM(88)] could not be detected in COS cells,and alternative constructs in which the stop codon was shifted toseveral sites N- and C-terminal to residue 88 also failed to producedetectable amounts of protein.

Transfected cells were cloned and individual clones secreting ICAM-1protein were identified by radioimmunoassay of culture supernatants. Theexpression of tICAMs was monitored by immunoprecipitation of[³⁵S]-cysteine-labeled culture supernatants with monoclonal orpolyclonal antisera followed by SDS-PAGE. Two monoclonal antibodies,c92.5 (which recognizes the same epitope as c78.4) and c78.5, define twodistinct conformational epitopes on ICAM-1. These two antibodies wereutilized in an RIA for soluble ICAM-1. c92.5 IgG was absorbed ontoIMMULON®-4 (microtiter plates, Dynex Technologies, Inc., Chantilly,Va.), the plates blocked with 10 mg/ml BSA, and the plates incubatedwith tICAM-containing samples. The plates were then washed, incubatedwith ¹²⁵I-c78.5 IgG, and the bound radioactivity determined. Theconcentration of tICAM was determined by comparison to a standard curveof purified ICAM-1.

Cell lines secreting tICAM(453) (CT.2A) and tICAM(185) (CD12.1A) wereselected for further study and were subjected to gene amplification inmethotrexate-containing media [Bebbington et al., supra]. In order toobtain sufficient quantities of protein for functional and structuralstudies, CHO cell transfectants were cloned and subjected to stepwisegene amplification in increasing concentrations of methotrexate. A clonederived from CT.2A (resistant to 100 nM methotrexate) and a clonederived from CD12.1A (resistant to 1 μM methotrexate) were used forpurification of soluble truncated proteins.

This resulted in the derivation of cell lines secreting 1.5 μg/ml oftICAM(453) and 1.0 μg/ml of tICAM(185). A stable cell line expressingtICAM(283) was not obtained, perhaps because the low level of secretionwas at the limit of sensitivity of the immunoassay. The cells wereadapted to serum-free media and the secreted ICAM-1 proteins purified tohomogeneity from culture supernatants (FIG. 2C).

tICAM(453) and tICAM(185) were purified from culture supernatants oftheir respective CHO transfectant cell lines by monoclonal affinitychromatography as described in Greve et al., Cell (1989) 56:839-847,followed by ion exchange chromatography on MONO-Q®(ion exchange resin,Pharmacia, Piscataway, N.J.) [for tICAM(453); absorption in 10 mM Tris(pH 6.0), elution in a 0-0.5 M NaCl gradient] or gel filtration onSUPEROSE®-12 (filtration gel, Pharmacia, Piscataway, N.J.) [fortICAM(185)] columns (Pharmacia). Protein was quantitated by amino acidanalysis and by RIA. Amino acid analysis was performed on an AppliedBiosystems Model 420A amino acid analyzer.

EXAMPLE 4 Structure of tICAMs

Blocking studies with the panel of six monoclonal antibodies to ICAM-1(all of which inhibit virus binding to ICAM-1) indicated that there aretwo distinct epitopes defined by these antibodies, one defined by c78.4(containing c78.1, c78.2, c92.1 and c92.5), and the other by c78.5.Immunoprecipitation studies with proteolytic fragments of ICAM-1 andwith in vitro translations of truncated ICAM-1 cDNAs indicate that bothof these epitopes are contained within the first Ig-like domain.

In vitro virus-binding studies utilizing radiolabeled tICAM(453) andpurified HRV have indicated that it can bind to rhinovirus in solution.

ICAM has been predicted, based on homology to NCAM, to be a member ofthe immunoglobulin gene superfamily. One would expect that theimmunoglobulin-like domains in ICAM would have the basic “immunoglobulinfold”, as has been shown for two other members of this family,beta-2-microglobulin and the HLA-A2 alpha-3 domain. This fold consistsof a “beta-barrel” conformation consisting of two antiparallelbeta-pleated sheets, one composed of three and one composed of four betastrands; a disulfide bond between two cysteine residues (separated byapproximately 60 amino acids along the chain) connects the two sheets[Williams, A. F., Immun. Today (1987) 8, 298-303]. Two of the disulfidebonds, those corresponding to domains II (C110-C161) and III(C212-C265), have been experimentally determined by us, providingsupport for the model. This model for the structure provides a basis fordesigning unique analogs that could mimic the virus-binding site and beuseful as receptor blockers. Each pair of antiparallel beta strands inthe beta-barrel is linked by a hairpin turn of variable size; such turnsor loops that protrude from secondary structures are often found to playroles in recognition of ligands [Lezczynski and Rose, Science (1986)224:849-855]. Such protruding structures may be of particular interestin the rhinovirus receptor, since the receptor-binding site on the viruscapsid is proposed to be in a recessed cavity. Using the sequence of theHRR, such turns and loops can be predicted based on a beta-barrelstructure and produced as synthetic peptides with addition of novelcysteine residues at the N- and C-terminus of the peptides; a disulfidebond can then be formed between such residues on the same peptide toclose the loop covalently (in contrast to the native protein, whereinthe loop would be closed by noncovalent interactions between theadjacent beta-strands). Such peptides would have a conformation moreanalogous to the conformation in the native protein than a simple linearpeptide, and can be tested for virus-binding activity.

EXAMPLE 5 Hydrodynamic Properties

The value f_(exp)/f_(o) of truncated ICAM-1 proteins were determinedfrom the apparent Stokes radii. (R_(s)) determined by gel filtration ona SUPEROSE®-12 column calibrated with protein standards (ferritin, 61.0A; catalase, 52.2 A; bovine serum albumin, 35.5 A; ovalbumin, 30.5 A;and RNase A, 16.4 A) and the calculated molecular weights of theproteins, using the value of M calculated from the core-glycosylatedform of the proteins, determined by synthesis in the presence ofswainsonin.

f _(exp)=6πηR _(s)

f_(o) (frictional coefficient of solvated sphere) was determined from:

f _(o)=(v ₂ +δv ₁ ^(o) /v ₂)^(⅓) f _(min)

where

f _(min)=6πη(3Mv ₂/4πN _(o))^(⅓)

(frictional coefficient of unsolvated sphere) The following values areassumed: v₂=0.73 cm²/g (partial specific volume of protein), v₁ ^(o)=1.0cm³/g (partial specific volume of solvent), d₁=0.35 g H₂O/g protein(solvation of protein), r=0.01 g/(cm.s) (viscosity of solvent), and v₁^(o)=1.0 cm³/g (specific volume of the solvent).

We have examined several of the physical properties of tICAM(453) andtICAM(185). Both proteins were quantitatively immunoprecipitated by twomonoclonal antibodies directed against two distinctconformation-dependent epitopes on ICAM-1, indicating that theseepitopes were contained within the first two domains and providingevidence that the purified proteins were correctly folded. Thefrictional ration, f/f_(o), the ratio between the observed andcalculated frictional coefficients shown below for both tICAM (453) andtICAM (185) indicates that both fragments of ICAM-1 are highlyasymmetric and elongated molecules.

PHYSICAL PROPERTIES OF tICAM(185) and tICAM(453) tICAM(185) tICAM(453)Mr¹ 27.2 kD 64.1 kD R_(s) ² 39 A 52 A f/f_(o) ³ 1.9 1.9 ¹Calculated forcore-glycosylated proteins. ²Determined by gel filtration; desialationof the proteins had no effect on the R_(s) values. ³Calculated asdescribed above.

The ratio of experimental and calculated frictional coefficients,f/f_(o), of both tICAMs indicate highly asymmetric and elonged shapes,and are consistent with the data reported for a species essentiallyidentical to tICAM(453) [Staunton, et al., Cell (1990) 61:243-254].Proteins belonging to the immunoglobulin supergene family would beexpected to have domains with the “immunoglobulin fold” motif, which isbasically two apposed sheets of antiparallel beta-strands withconnecting loops. In support of the immunoglobulin homology, disulfidemapping studies of ICAM-1 have revealed the existence of intradomaindisulfide bonds predicted by the immunoglobulin fold in domains 1-4.This is consistent with the data of Staunton, et al., Cell (1988)52:925-933 with regard to tICAM(453).

EXAMPLE 6 Circular Dichroism

CD spectra were recorded on an AVIV model 62DS spectrometer. Proteinsolutions at approximately 0.5 mg/ml (determined by amino acid analysisin the indicated buffers) were scanned at 20 C. in a cell with a 0.1 cmpathlength. Five respective scans (1 nm interval, 1.5 nm bandwidth) wereaveraged and buffer-subtracted for each spectrum.

Circular dichroism (CD) spectra were obtained for the soluble forms ofICAM. A single minima at 210-220 nm is indicative of the presence ofbeta-structure and should be seen in proteins containingimmunoglobulin-like domains because of the extensive amount ofbeta-structure in the “immunoglobulin fold” (Williams, A. F., and A. N.Barclay, Ann. Rev. Immunol. (1988) 6:381-405. As internal standards,spectra were collected for beta-2-microglobulin and a monoclonal IgG,immunoglobulin-like proteins of known three-dimensional structure[Becker, J. W. and G. N. Reeke, Proc. Natl. Acad. Sci. USA (1985)82:4225-4229; Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett,J. L. Strominger, and D. C. Wiley, Nature (1987) 329:506-512; Amzel, L.M. and R. J. Poljak, Ann. Rev. Biochem. (1979) 48:961-967]. As expected,the spectra of beta-2-microglobulin (not shown) and IgG (FIG. 3A)possessed single major minima at 215 nm and 217 nm, respectively,indicative of extensive amounts of beta-structure. The spectra of thetwo truncated proteins tICAM(453) and tICAM(185) were similar to eachother and to that of ICAM-1 (FIGS. 3B, C, and D), with minima at 216-217nm and a broad shoulder at 225-230 nm, although the shoulder was moreprominent with tICAM(185) than tICAM(453). Considered qualitatively,these CD spectra provide evidence that the soluble ICAM-1 proteins arefolded and possess considerable amounts of beta-structure. The shoulderat 225-230 nM present in the three tICAMs is not found in the spectra ofbeta-2-microglobulin and IgG, suggesting the presence of secondarystructural features not present in proteins with “classical”immunoglobulin-like domains. The fact that the shoulder at 225-230 ismore prominent in tICAM(185) suggests that the secondary structuralfeatures detected by this method are localized in the first two domains.These CD data are similar to those reported by Sayre, P. H., R. E.Hussey, H-C Chang, T. L. Ciardelli, and E. L. Rheinherz, “Structural andbinding analysis of a two domain extracellular CD2 molecule,” J. Exp.Med. (1989) 169:995-1009] for a soluble form of CD2 (which is alsothought to be a member of the immunoglobulin supergene family) in thatsignificant amounts of alpha-helix (which is essentialy absent fromimmunoglobulin molecules) was predicted from the CD spectra. Whenindividual domains of ICAM-1 are compared to domains from other membersof the immunoglobulin supergene family by the ALIGN program [Dayoff, M.O., W. C. Barker, and L. T. Hunt, Methods in Enzymology (1983)91:524-545], the similarity is quite limited, with only domains II andIII of ICAM-1 having significant scores (above 3 SD; for discussion seeWilliams, A. F., and A. N. Barclay, Ann. Rev. Immunol. (1988)6:381-405]. Domain I has a significant score only when compared withdomain I from ICAM-2 and VCAM-1, proteins that are closely related infunction. In addition, domain I has a number of unusual features, suchas a short distance (44 residues) between intradomain disulfide bondsand the presence of four cysteines instead of the usual two in the B andF beta-strands.

EXAMPLE 7 Radioactive Labeling of tmICAM-1, tICAM(185), and tICAM(453)and Demonstration of Retained Capacity for Binding to MonoclonalAntibodies

The epitopes reactive with monoclonal antibodies c78.4A and c78.5A areconformationally-dependent epitopes and thus can be used an analyticalprobes for confirming retention of the native ICAM structure. Knownamount of purified ICAM were incubated with c78.4A or c78.5AIgG-SEPHAROSE® and the fraction of the radioactivity bound determined.These experiments showed that the purified tmICAM-1, tICAM(185), andtICAM(453) completely retained the ability to bind to these monoclonalantibodies.

Transfectants were metabolically labeled with [³⁵S]-cysteine, and celllysates (for transmembrane ICAM) or culture supernatants (for truncatedICAM) were prepared and incubated with c78.4A IgG-SEPHAROSE® beads. Thebeads were washed and absorbed proteins were eluted with SDS andanalyzed by SDS-PAGE [see Greve, et al., Cell (1989) 56:839-847]. It wasfound that the isolated proteins were quantitatively bound to the c78.4Aand c78.5A Mabs.

Accordingly, the tICAM(185) and tICAM(453) both have retained nativeICAM structure.

EXAMPLE 8 Human Rhinovirus Binding Assays of Transmembrane and ofNon-Transmembrane Truncated Forms of ICAM-1

Described below are three binding assays used to assess binding activityof the various forms of ICAM.

A. Pelleting Assay

[³⁵S]-Cysteine-labeled tmICAM-1 or tICAM was mixed with HRV3 in 100 ulof 10 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 0.1%Triton X-100. The mixture was incubated for 30 minutes at 37° C., cooledon ice, layered on top of a cushion of 200 μl of 10% glycerol, 0.2 Mtriethanolamine (pH 7.5), and centrifuged in a Beckman air-drivencentrifuge at 134,000×g for 30 minutes at 4° C. The top 275 μl wasremoved, and the pellet was analyzed by SDS-PAGE and scintillationcounting. Silver-staining of SDS gels of control experiments indicatedthat essentially all of the HRV3 is pelleted under these conditions andessentially all of the ICAM remains in the supernatant. The results areshown below:

ICAM % ICAM Pelleted¹ tmICAM-1 11.6% tICAM(453) 1.0% tICAM(185) 4.3%¹Average of 4 experiments; these numbers cannot be directly convertedinto relative affinities.

These data show that both truncated forms of ICAM bind to rhinovirus.

B. Solution Binding Assay

Previous work has demonstrated that detergent-solubilized tmICAM-1 canbind to rhinovirus [Greve, et al., Cell (1989) 56:839-847] and that theextracellular domain of ICAM-1 competes for rhinovirus binding to cells[Marlin, et al., Nature (1990) 344:70-72]. To quantitatively compare thevirus-binding properties of the soluble ICAM-1 proteins, a solid-phasecompetition assay was employed. In these experiments, varyingconcentrations of soluble competitor ICAM-1 proteins were assayed forthe ability to compete for the binding of [³⁵S]-HRV3 to immobilizedICAM-1; nonionic detergent TRITON® X-100 (nonionic surfactant, UnionCarbide, Wilmington, Del.) was included in the buffers so that thedifferent proteins could be compared under identical conditions. First,tmICAM-1 (isolated in the presence of 0.1% octylglucoside instead ofTRITON® X-100) was diluted 10-fold into a Tris/NaCl buffer and allowedto adsorb to the walls of a microtiter plate (IMMULON®-4, Dynatech)overnight. Nonspecific binding sites on the plate were then blocked with10 mg/ml BSA and binding experiments performed in 0.1% TRITON® X-100/1mg/ml BSA/Tris/NaCl. Approximately 20,000 cpm of [³⁵S]-HRV3 were mixedwith varying amounts of tmICAM, tICAM(453) or tICAM(185); incubated for1 hour at 37° C., and then added to wells of the microtiter plates andincubated for 3 hours at 37° C. The plates were washed and the boundradioactivity determined.

As shown below, tmICAM-1 inhibits virus binding half-maximally at lowconcentrations (0.008 μM) while tICAM(453) and tICAM(185) inhibit atmuch higher concentrations (3.1 μM and 9.7 μM, respectively; or 350- toalmost 1,000-fold higher than tmICAM).

ICAM IC₅₀ ¹ tmICAM 8.0 ± 3.3 nM tICAM (453) 3.1 ± 1.8 uM tICAM (185) 9.7± 3.2 uM ¹Concentration of ICAM-1 protein needed to inhibit [³⁵S]-HRV3binding to ICAM-1-coated microtiter plates by 50%; average of threeexperiments ±S.D.

These data confirm and extend the earlier observations that tICAM(453)and tICAM(185) do bind to rhinovirus but with lower affinities than doestmICAM-1 and provide evidence that the virus-binding site is encompassedwithin the two N-terminal domains (first 185 residues) of ICAM-1. TheIC₅₀ value of tICAM(453) is comparable to that of tICAM(185). The small(three-fold) lower IC₅₀ of tICAM(185) is reproducible but of unknownsignificance at present. It has been reported that a mouse-human chimeraof transmembrane ICAM-1 in which the first two domains are of humanorigin is capable of binding rhinovirus as well as human ICAM-1, while ashortened transmembrane form containing only the two N-terminal domainsbinds rhinovirus at approximately one-tenth the level of full-lengthICAM-1 [Staunton, et al., Cell (1990) 61:243-254]. These workersinterpret these results as demonstrating that domains I and II areimportant for rhinovirus binding while the distance from the plane ofthe membrane determines the efficiency with which the rhinovirus canapproach the binding site. The interpretation of these results islimited by the requirement for species-specificity of all possibleinteractions with the rhinovirus and by the steric limitations ofshortened transmembrane molecules. Our results with the soluble ICAM-1clearly and quantitatively demonstrate that the virus-binding site iscontained within the first two N-terminal domains.

C. Dot-Blot Assay

An alternative method of measuring binding activity was utilized inwhich tmICAM-1, tICAM(453), or tICAM(185) were adsorbed tonitrocellulose filters, the non-specific binding sites on the filtersblocked with 10 mg/ml bovine serum albumin (BSA), and radioactive virusor ¹²⁵I-monoclonal antibody to ICAM-1 incubated with the filter for 60minutes at 37° C. The filters were washed with buffer and the filtersexposed to X-ray film.

The amount of radioactivity bound to the filters was determined bydensitometry of the autoradiograms, and the data is expressed as HRV3binding (in arbitrary units) normalized to the amount of ICAM bound tothe blot by a parallel determination of the amount of ¹²⁵I-monoclonalantibody c78.4A or c78.5A bound to the ICAM (bound to the blot). Theresults are shown below:

Binding of [³⁵S]-HRV3 to Immobilized ICAM¹ ICAM tICAM (453) ratioICAM/tICAM (453) 1.2 ± 1.1 0.52 ± 0.45 2.3 ¹Average of 5 experiments.Data is expressed in arbitrary densitometric units of [³⁵S]-HRV3binding/[¹²⁵I]-anti-ICAM monoclonal antibody binding.

The results from this experiment indicate that under these assayconditions tICAM(453) is capable of binding rhinovirus at levelscomparable to those of tmICAM-1 when the amount of virus bound wasnormalized to the amount of [¹²⁵I]-Mab bound. Further, these resultsindicate that the tICAM forms are capable of binding to rhinovirus, butthat the binding avidity is dependent to some degree upon theconfiguration of the tICAM. tmICAM-1 may be a small multimer (probably adimer) and presentation of tICAM in a multimeric form mimics thismultimeric configuration.

Evidence supporting this hypothesis comes from quantitative bindingstudies, in which the ratio of the maximum number of rhinovirusparticles and the maximum number of antibody molecules that can be boundto cells is approximately 1.5. This is in contrast to the earlier workof Tomassini, J., et al., (1986) J. Virol. 58:290, which suggested acomplex of five molecules needed for binding. Their conclusion was basedon an erroneous interpretation of gel filtration data that failed totake into account bound detergent molecules.

EXAMPLE 9 Infectivity Assays

HRVs and [³⁵S]-HRV3 was propagated and purified as described in Example1). All infectivity assays were performed with HeLa-S3 in Dulbecco'sModified Essential Medium/2% fetal calf serum. Assay I: 0.1 ml ofdilutions of HRV3 (with 10⁴-10⁻¹ pfu/ml) in the presence or the absenceof tICAM(185) or tICAM(453) were added to 10 wells of a 96-wellmicrotiter plate containing 2×10⁴ HeLa cells/well in 0.1 ml. Thecultures were incubated for five days at 34° C. and the titer determinedfrom the number of infected wells at the limiting dilution. Assay II:10⁷ PFU of HRV3 and various concentrations of ICAM-1 in a final volumeof 25 μL of 10 mM HEPES (pH 7.5)/150 mM NaCl were incubated for 30minutes at 37° C., and then serially diluted into culture medium. Thevirus was then incubated with HeLa cells at 10⁶ cells/ml for 30 minutesat room temperature, plated into 10 wells of a 96-well microtiter plateat 10⁵ cells/well, and then scored for infectivity as described above.Assay III: 0.1 ml of HRV3 (0.1 μg/ml, 10⁷ PFU/ml) was preincubated for30 minutes at 37° C. with ICAM-1 and then added to wells of a 96-wellmicrotiter dish containing 10⁵ HeLa cells and incubated for 24 hours.The cultures were then scored by staining with crystal violet [Minor, P.D., in Virology: A practical approach, B. W. J. Mahy, ed. (IRL Press,Oxford, 1985), pp. 25-40] and determining the optical density at 550 nm.All experiments were performed in triplicate and the results expressedat the concentration of ICAM-1 needed to reduce the OD₅₅₀ by 50%.

The effect of soluble ICAM-1 on HRV infectivity was examined in threedifferent infectivity assays, the first two being limiting dilutiondeterminations and the third being a high multiplicity of infection(MOI) experiment using the same virus concentrations as the virusbinding assay in Example 16. In assay I, cells were infected with serialdilutions of HRV3 in the continuous presence of various concentrationsof ICAM-1 and the reduction in virus titer determined. As shown below,the IC₅₀ of tICAM(185) for neutralization is 8.2 μM, similar to theconcentration needed to inhibit virus binding. In contrast, the IC₅₀ forneutralization of tICAM(453) is 0.38 μM, 10-fold lower than theconcentration needed to block virus-receptor binding:

NEUTRALIZATION OF RHINOVIRUS BY SOLUBLE ICAM-1 IC₅₀ ¹ tICAM(185)tICAM(453) tmICAM-1 Assay I (continuous) 8.2 μM 0.38 μM ND Assay II(pretreatment) <20 μM <20 μM 0.03 Assay III (high MOI) 13.2 μM 1.2 μM ND¹Concentration of ICAM-1 protein needed to inhibit HRV3 infectivity by50%, as described for each assay above.

Thus, the neutralizing activity of tICAM(185) is directly correlatedwith its effect on virus receptor binding, while tICAM(453) isneutralizing HRV at a considerably lower concentration and is presumablyacting by a mechanism other than direct competition for receptor-bindingsites on the virus. Assay II consists of preincubating HRV3 with ICAM-1,serially diluting the mixture into culture medium, and then adding thevirus to the cells; under these conditions the ICAM-1 is diluted out tonegligible concentrations for the actual infection. tICAM(185) andtICAM(453) had essentially no neutralizing activity in this assay atconcentrations as high as 20 uM protein, indicating that the effects ofthe soluble ICAMs on rhinovirus must be reversible. Assay III consistsof a single cycle infection at an MOI of 10 in the continuous presenceof various concentrations of soluble ICAM-1. Under these conditionstICAM(453) neutralized HRV with an IC₅₀ of 1.2 μM, an 11-fold lowerconcentration than that of tICAM(185). The relative difference in theconcentrations of the two proteins needed to neutralize rhinovirus inAssay III is essentially the same as in Assay I, although the absoluteconcentrations are approximately 3-fold higher, perhaps because of anonlinear relationship between infectious particles and infected cellsunder conditions of high MOI.

The neutralization activities of the two soluble ICAM-1 species examinedhere are intriguing in that they indicate multiple mechanisms of virusneutralization by receptor. The first mechanism, exemplified bytICAM(185), appears to be a simple competitive inhibition ofvirus-receptor binding by soluble receptor. The second, exemplified bytICAM(453), involves neutralization of virus at a concentration belowthat necessary to inhibit virus-receptor binding. This disparity betweenthe virus-neutralizing and virus-binding activities of tICAM(453)indicates that steps in virus entry and/or uncoating are affected bythis protein. These apparently different mechanisms of virusneutralization may be reflecting different steps in the entry anduncoating process in which the membrane-bound receptor participates in anormal infection. The modes of action of the capsid-binding WINcompounds as revealed by their effects on different rhinovirus serotypes[Pevear, D. C., M. J. Fancher, P. J. Felock, M. G. Rossman, M. S.Miller, G. Diana, A. M. Treasurywala, M. A. McKinlay, and F. J. Dutko,J. Virol. (1989) 63:2002-2007], where some serotypes are blocked frombinding to the receptor while other serotypes are blocked at anintracellular uncoating step, suggest a similar “hierarchy” of sites atwhich the infection can be interrupted. Both of the neutralizationmechanisms mediated by soluble ICAM-1 are reversible, sinceneutralization is seen when cells are infected in the continual presenceof soluble ICAM but not when the virus is preincubated at comparableconcentrations of ICAM-1 followed by dilution of the ICAM-1 tonegligible concentrations. During many picornavirus infections,noninfectious “altered” virions can be recovered from cells, presumablyas a direct or indirect result of interaction with the receptor[Roesing, T. G., P. A. Toselli, and R. L. Crowell, J. Virol. (1975)15:654-667; Guttman, N. and D. Baltimore, Virol. (1977) 82:25-36]. Therelationship of the receptor-neutralized rhinoviruses to such “altered”virions found in other picornaviruses is unclear at present.

Marlin, et al., Nature (1990) 344:70-72 have reported that theextracellular domain of ICAM-1-neutralized virus at a concentration ofICAM considerably lower than that needed to inhibit virus-receptorbinding, although this discrepancy was attributed to differences invirus concentrations between the binding and infectivity two assays. Ourresults, which show a differential effect of the two soluble ICAM-1proteins even at virus concentrations identical to that in thevirus-binding assay indicate that there is a real difference betweenbinding and neutralizing properties of tICAM(453). Possible explanationsinclude: (1) cooperative interactions between ICAM-1 molecules at thesurface of the virus leading to more effective virus inactivation, (2)effects of the size of the ICAM-1/rhinovirus complex on virusinternalization or uncoating, or (3) interactions of portions of theICAM-1 molecule other than the two N-terminal domains with therhinovirus. In an interesting parallel in another system, the solubleCD-4 mediated neutralization of HIV, a nonlinear relationship betweensoluble CD4 concentration and neutralization at high fractionaloccupancy of gp 120 molecules was observed, providing evidence forpositive cooperativity between virus-bound CD4 molecules [Layne, S. P.,M. J. Merges, M, Dembo, J. L. Spouge, and P. L. Nara, Nature (1990)346:277-279]. While the molecular basis of tICAM(453)-mediatedneutralization or its significance to interaction of the rhinovirus withICAM-1 at the cell surface remains to be determined, it clearlyindicates a role for the three domains C-terminal to residue 185.

EXAMPLE 10 Cellular Adhesion Assay

JY cells at 10⁷ cells/ml (labeled with 10 μCi/ml [³⁵S-cysteine for 18hours) were preincubated with soluble ICAM-1 or indicated monoclonalantibodies for 30 minutes at 37° C. in Dulbecco's Modified EssentialMedium/2% fetal calf serum and then added to microtiter plates coatedwith ICAM-1. The plates were then incubated for 60 minutes at 37° C.,the plates washed three times with media, and then the bound cellsquantified by scintillation counting.

Soluble ICAM molecules were then examined for their ability to inhibitthe adhesion of JY lymphoblastoid cells (which express LFA-1) toICAM-1-coated tissue culture plates. As shown in FIG. 4, tICAM(185) andtICAM(453) both inhibited JY cell binding at identical concentrations of10 μM, indicating that the LFA-1 binding site is entirely encompassedwithin the first two domains of ICAM-1.

The identical inhibitory activities of tICAM(453) and tICAM(185) towardICAM-1/LFA-1-mediated cell adhesion clearly demonstrate that the LFA-1binding site is completely encompassed within the first 185 residues ofICAM-1. Previous work has indicated that the two N-terminal domains ofICAM-1 were important in LFA-1 binding, in that site-directedmutagenesis at a number of positions in domains I and II reduce theability to bind to LFA-1 and a shortened transmembrane moleculecontaining domains I and II retains one-third of the LFA-1 bindingactivity of full-length ICAM-1 [Staunton, et al., Cell (1990)61:243-254]. The relatively high concentration of soluble ICAM-1 neededto inhibit cell adhesion illustrates the fact that ICAM-1/LFA-1-mediatedcell adhesion is a cooperative result of multiple weak interactions, asmight have been predicted from the strong dependence of ICAM-1/LFA-1mediated cell adhesion on the density of ICAM-1 [Dustin, M. L. and T. A.Springer, J. Cell Biol. (1989) 107:321-331]. A micromolar dissociationconstant has been reported for the CD2/LFA-3 interaction (Sayre, P. H.,R. E. Hussey, H-C. Chang, T. L. Ciardelli, and E. L. Rheinherz,“Structural and binding analysis of a two domain extracellular CD2Molecule,” J. Exp. Med. (1989) 169:995-1009], suggesting that lowadhesion molecule affinity is a general phenomenon, perhaps to allow fortight regulation of the adhesion process by control of copy number. Theidentical inhibitory activities of both soluble ICAM-1 proteins towardcell adhesion provide additional evidence that both proteins arecorrectly folded and underscores the fact that the enhanced neutralizingactivity of tICAM(453) relative to tICAM(185) is specific toICAM-1/rhinovirus interaction.

While the present invention has been described in terms of specificmethods and compositions, it is understood that variation andmodifications will occur to those skilled in the art upon considerationof the present invention.

For example, it is anticipated that smaller protein fragments andpeptides derived from ICAM-1 that still contain the virus-binding sitewould also be effective.

Further, it is anticipated that the general method of the invention ofpreparing soluble protein forms from insoluble, normally membrane boundreceptor proteins can be used to prepare soluble multimeric forms ofother receptor proteins useful for binding to and decreasing infectivityof viruses other than those that bind to the “major group” receptor.Such other viruses include Herpes simplex and Epstein-Barr viruses.

Accordingly, it is intended in the appended claims to cover all suchequivalent variations which come within the scope of the invention asclaimed, and consequently only such limitations as appear in theappended claims should be placed thereon.

5 49 bases nucleic acid single stranded linear Other nucleic acid no nonot provided HindIII site bases 8-13 D.,Marlin,S.,Stratowa,C.,Dustin,M.,Springer,T.Staunton Primary structure of ICAM-1 demonstrates interactionbetween members of the immunoglobulin and integrin supergene familiesCell 52 925-933 25-MAR-1988 1 FROM 14 TO 49 1 GGAATTCAAG CTTCTCAGCCTCGCT ATG GCT CCC AGC AGC CCC CGG CCC 49 Met Ala Pro Ser Ser Pro Arg Pro5 40 bases nucleic acid single stranded linear Other nucleic acid no yesnot provided Pst1 site bases 8-13 D.,Marlin, S.,Stratowa,C.,Dustin,M.,Springer,T.Staunton Primary structure of ICAM-1 demonstrates interactionbetween members of the immunoglobulin and integrin supergene familiesCell 52 925-933 25-MAR-1988 2 FROM 17 TO 40 2 GGAATTCCTG CAGTCACTCATACCGGGGGG AGAGCACATT 40 40 bases nucleic acid single stranded linearOther nucleic acid no yes not provided BamHI site bases 8-13D.,Marlin,S.,Stratowa,C.,Dustin,M., Springer,T.Staunton Primarystructure of ICAM-1 demonstrates interaction between members of theimmunoglobulin and integrin supergene families Cell 52 925-93325-MAR-1988 3 FROM 17 TO 40 3 TTCTAGAGGA TCCTCAAAAG CTGTAGATGGTCACTGTCTG 40 39 bases nucleic acid single stranded linear Other nucleicacid no yes not provided BamHI site bases 8-13D.,Marlin,S.,Stratowa,C.,Dustin, M., Springer,T.Staunton Primarystructure of ICAM-1 demonstrates interaction between members of theimmunoglobulin and integrin supergene families Cell 52 925-93325-MAR-1988 4 FROM 17 TO 39 4 TTCTAGAGGA TCCTCAAAAG GTCTGGAGCT GGTAGGGGG39 39 bases nucleic acid single stranded linear Other nucleic acid noyes not provided BamHI site bases 8-13D.,Marlin,S.,Stratowa,C.,Dustin,M., Springer,T.Staunton Primarystructure of ICAM-1 demonstrates interaction between members of theimmunoglobulin and integrin supergene families Cell 52 925-93325-MAR-1988 5 FROM 17 TO 39 5 TTCTAGAGGA TCCTCACCGT TCTGGAGTCC AGTACACGG39

We claim:
 1. In a method for neutralizing human rhinovirus of the majorreceptor class by contacting said rhinovirus with a soluble form ofICAM-1, the improvement comprising contacting said virus withtICAM(453).
 2. In a method for reducing the infection by humanrhinovirus (HRV) of a host cell susceptible to infection by HRV bycontacting the virus with tICAM(453) under conditions favorable forbinding, the improvement wherein said tICAM(453) is at a concentrationbelow that required for inhibition of binding of said HRV to said hostcell.